Inadequate adherence to HIV pre-exposure chemoprophylaxis (PrEP) and antiretroviral therapy (ART) is common, and the leading cause of therapeutic failure. Despite this damaging impact on human health, reliable, convenient methods with which to quantify adherence routinely are not available. This application proposes a new quantitative approach to routinely monitor adherence to tenofovir disoproxil fumarate (TDF)-based PrEP and ART. TDF-based regimens are the cornerstone of PrEP and ART with approximately 80% of ART patients and 100% of PrEP patients receiving TDF-based therapy. We discovered that tenofovir-diphosphate (TFV-DP), the active form of TFV, has a 17 day half-life in red blood cells (RBC) with low variability. These characteristics are analogous to glycosylated hemoglobin A1C, which is measured as standard- of-care to quantitatively monitor cumulative glucose exposure for diabetes. We propose that the level of TFV-DP in RBC can be used in a similar way to quantitatively monitor cumulative drug exposure (adherence) for TDF-based PrEP and ART. We showed that the level of TFV-DP in RBC can be quantified using dried blood spots (DBS), which offers significant advantages such as low expense and easy collection and processing requirements. The proposed study will determine the effect of adherence rate on the level of TFV-DP in DBS. Forty eight HIV-negative volunteers will be randomized to 33%, 67% and 100% of daily dosing with TDF-FTC. All dosing will be directly observed therapy (DOT). A randomized incomplete block design with a washout period will be employed such that participants receive 2 of 3 dosing regimens for 12 weeks each. A separate cohort of 32 HIV-positive volunteers will receive daily DOT with TDF-FTC-based therapy. Expected levels of TFV-DP in DBS in the setting of DOT will be defined, compared in HIV-positive versus negative persons, and used to construct a model that predicts adherence quantitatively for PrEP and ART. We will immediately translate our findings to interpret TFV-DP in DBS that are currently being collected for several pivotal PrEP demonstration projects where adherence monitoring is a key outcome. We believe that TFV-DP in DBS, as a new quantitative measure of adherence, has the potential to make an immediate and lasting public health impact.